Characterization of transgenic tomato plants expressing an antisense catalase gene and cloning of a TOMCAT2 gene
Abstract
Transgenic tomatoes (Lycopersicon esculentum Mill. ‘Ohio 8245’) expressing an antisense catalase gene (ASTOMCAT1) were used to test the hypothesis that modification of the reactive oxygen species scavenging mechanism in plants can lead to changes in oxidative strew tolerance. A two to eight-fold reduction in total catalase activity and a two-fold increase in levels of H2O2 were detected in the leaf extracts of transformants. Electrophoretic characterization of multiple catalase isoforms revealed the specific suppression of TOMCAT1 in transgenic plant. Transgenic plants did not survive 4°C chilling stress and transgenic plants treated with 3% H2O2 showed visible damage within 24 hours and subsequently died. In contrast, wild-type and azygous control plants recovered from the treatment. Transgenic antisense catalase fruit exhibited low catalase activity and mRNA level during fruit ripening which was correlated with a delay in ethylene production and fruit ripening. A loss of catalase led to elevated hydrogen peroxide and an increase in ascorbate peroxidase activity in the mature-green stage of antisense tomato fruit. Despite the compensation of alternative H2O2 scavenging pathway by ascorbate peroxidase, suppression of catalase activity in transgenic tomato resulted in enhanced sensitivity to low temperature stress in fruit. These results support a role of catalase in oxidative stress defense system in tomato. A novel full length cDNA clone for tomato catalase transcript (pTOMCAT2) was isolated and characterized along with pTOMCAT1. Both pTOMCAT1 and pTOMCAT2 sequences contain the conserved Ser-Arg-Leu peroxisomal targeting sequence. RNA gel blot analysis with gene specific probes reveals that TOMCAT1 and TOMCAT2 exhibited different expression patterns during development and responded differently to various biotic and abiotic: signals. The expression of antisense RNA of TOMCAT1 had a slight effect on TOMCAT2 gene expression in homozygous transgenic tomato plants. These transgenic tomato plants are capable of maintaining elevated levels of H2O2 resulting in an increased resistance to virulent strains of Pseudomonas syringes pvtomato and an induction of PR genes. Our development of transgenic tomato plants lacking endogenous catalase activity and elevated in H2O 2, provides a valuable tool for the dissection of the processes thought to be mediated by ROSs.
Degree
Ph.D.
Advisors
Woodson, Purdue University.
Subject Area
Molecular biology|Botany|Plant propagation|Genetics
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